Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC

Harunori Yoshikawa, Mark Larance, Dylan J. Harney, Ramasubramanian Sundaramoorthy, Tony Ly, Thomas Owen-Hughes, Angus I. Lamond (Lead / Corresponding author)

Research output: Contribution to journalArticle

2 Citations (Scopus)
110 Downloads (Pure)

Abstract

We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.

Original languageEnglish
Article numbere36530
Pages (from-to)1-26
Number of pages26
JournaleLife
Volume7
Issue number7
DOIs
Publication statusPublished - 10 Aug 2018

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Keywords

  • Amino Acids/chemistry
  • Animals
  • Cell Line
  • Chromatography, High Pressure Liquid
  • Humans
  • Mice
  • Polyribosomes/chemistry
  • Protein Biosynthesis
  • Proteomics
  • Ribosomes/chemistry

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